Voltage-gated Nav channel Nav1.5 (encoded by SCN5A) initiates rapid depolarization of the cardiac action potential and is essential for normal cardiac conduction. Human SCN5A mutations may lead to cardiac arrhythmia and sudden death. Nav1.5 function is determined by its channel properties as well as its cellular localization. The identity of the cellular pathway(s) required for Nav1.5 localization at excitable membranes in heart is an important and currently unresolved question. Our long-term goals are to elucidate the cellular pathway(s) and molecular determinants underlying cardiac Nav1.5 targeting. Our specific hypothesis is that ankyrin-G (a membrane adaptor protein) is required for Nav1.5 targeting to intercalated disc and T-tubule membrane domains. We base this hypothesis on previous observations that 1) targeted knockout of ankyrin-G in mouse cerebellum blocks targeting of Nav1.6 and 1.2 in neurons, 2) ankyrin-G binds Nav1.2 through a 9 residue motif on Nav1.2 loop 2, 3) this motif is required Nav1.2 targeting in neurons, and 4) Nav1.5 contains a nearly identical sequence in loop 2. Additionally, our preliminary results support the interaction and co-localization of Nav1.5 and ankyrin-G in heart, and suggest that Nav1.5 requires ankyrin-G-binding for targeting and normal physiological function in humans. Based on these observations, the experiments in this proposal will test a role for an ankyrin-G- based pathway for Nav1.5 targeting in heart. We predict that these experiments will supply the first evidence for a cellular pathway required for cardiac Nav1.5 targeting, and provide in vivo evidence for a new class of human Na 'channelopathies' due to abnormal Nav1.5 targeting. The specific aims are to: 1) Determine the structural requirements for ankyrin-G/Nav1.5 interactions and test human Nav1.5 (SCN5A) disease mutants for ankyrin-G loss-of-binding. 2) Evaluate the requirement of ankyrin-G for Nav1.5 expression, targeting, and function in heart. 3) Characterize the ankyrin-G pathway for Nav1.5 targeting in cardiomyocytes including identification of cellular intermediates in Nav1.5 targeting pathway and identification/characterization of ankyrin-G-interacting proteins for effects on ankyrin-G/ Nav1.5 localization and expression.